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Orientational Imaging of Single Gold Nanorod at Liquid/ solid Interface with Polarized Evanescent Field Illumination Lin Wei, Jianghong Xu, Zhongju Ye, Xupeng Zhu, Meile Zhong, Wenjuan Luo, Bo Chen, Huigao Duan, Quanhui Liu, and Lehui Xiao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04695 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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Analytical Chemistry

Orientational Imaging of Single Gold Nanorod at Liquid/solid Interface with Polarized Evanescent Field Illumination Lin Wei,†,‡ Jianghong Xu,†,‡ Zhongju Ye,‡ Xupeng Zhu,§ Meile Zhong,‡ Wenjuan Luo,‡ Bo Chen,‡ Huigao Duan,§ Quanhui Liu,§ and Lehui Xiao*,‡ ‡

Dynamic Optical Microscopic Imaging Laboratory, Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410082, People’s Republic of China. § School of Physics and Electronics, Hunan University, Changsha, 410082, People’s Republic of China

Supporting Information Placeholder ABSTRACT: Understanding the mechanistic information of many kinetic processes requires the exploration of dynamic rotational information of the target object at single particle (or molecule) level. In this work, we developed a new strategy, total internal reflection scattering (TIRS) microscopy, to determine the full three-dimensional (3D) angular information of single gold nanorod (GNR) close to the liquid/solid interface. It was found that the 3D orientational information of individual GNR could be readily elucidated by using p-polarized TIRS illumination through deciphering the orientation-coded intensity distribution pattern in single TIRS image. In comparison with the previously reported strategies, this method doesn’t require complicated focal plane correction, affording a versatile pathway to track the rotational dynamics close to the interface in a high throughput manner. The methodology presented here, therefore, demonstrates a promising approach that can be applied to fluidic membranes, including membranes with polymers, bound proteins and so on.

Probing dynamics of individual entities by optical means is at the peak of demand for the exploration fundamental mechanisms of physical, chemical and biological processes in nanoscale system.[1] The mechanistic information of many interesting kinetic processes has been resolved based on the spatial localization information such as the receptor clustering process on cell membrane, the cellular infection pathway of virus and so on.[2] However, besides the positional information, for the rational comprehending of the underline mechanistic information of many complex kinetic processes, it is fundamentally important to elucidate the orientational information real-time. Examples include the rotational mechanism of F1-ATPase,[3] the conformation change of dynamin during the endocytosis process,[4] modification of a dipole emission close to a nanostructure as well as the conformation change of polymers near the glass transition temperature.[5] Polarization sensitive optical imaging based on fluorescent signal has been widely applied to probe the local orientation of discrete entities.[6] Owing to the weak stability of the fluorescent probe against photo-bleaching and the drawback of photoblinking, the requirement of high accuracy and long-term observations for complex biological processes becomes a grand challenge. The development of nanometer-sized gold and silver colloids has produced a new class of biological labels.[7] Particularly, for

anisotropic nanoparticle, the surface plasmon is not evenly distributed around the nanoparticle, manifesting in shape and orientational dependence of extinction cross-section. One of the beststudied anisotropic nanoparticles is GNR, which exhibits longitudinal and transverse surface plasmon modes with oscillation direction parallel to the long and short axes, respectively. Based on the orientation dependent surface plasmon resonance effect, several interesting methods have been developed to determine the angular information of single GNR, such as cross polarization imaging,[3b,8] photothermal imaging and differential interference detection.[9] These methods have been adopted to resolve the rotational dynamics of individual GNR in complex environments. One of the major limitations of these polarization detection schemes is that the results only reflect the orientation information projected onto the x-y plane within the first quadrant. It is thus difficult to distinguish the concrete angular information from the symmetric positions in the Cartesian coordinate system. In addition, the angular fluctuations along z direction are hardly resolvable just based on the polarization anisotropic value. To address these limitations, in this work, we demonstrated a new and convenient strategy to determine the full three-dimensional angular information of GNR close to the solid/liquid interface with TIRS microscopy. By tuning the excitation wavelength to the longitudinal resonance mode, the selectivity and sensitivity toward the rod shape plasmonic probe were greatly enhanced. Only the probe near the interface could be excited by the evanescent field.[10] More interestingly, enhancement of scattering from the vertical electric dipole relative to the horizontal electric dipole was observed by using a p-polarized light illumination. The observed scattering image of individual GNR at the focal plane exhibited characteristic doughnut shape, reminiscent of the magnified point spreading function (PSF) from the defocused dark-field image. The angular information of the GNR could thus be determined by deciphering the orientation coded characteristic intensity distribution pattern in the TIRS image. Distinct from the defocused illumination mode,[11] the nanoparticle is still located in the focal plane. The shape of the diffraction-limited PSF from the GNR could then be well manipulated by tuning the polarization direction of the light for either rotational diffusion imaging or high-accuracy spatial localization tracking.

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Figure 1. a) Schematic diagram of the setup for TIRS imaging. b) Schematic representation of the polarization dependent TIRS image of GNR at the liquid/solid interface. c) FDTD simulation of the polarization dependent electric field distribution of a GNR located at the liquid/solid interface with the polar angle of 10o and azimuthal angle of 90o.

The single particle imaging experiments were conducted on a home built TIRS microscope, Figure 1.[12] For the efficient excitation of the probe at the interface, the incident laser line was selected to match the resonance peak of GNR and directed to the side of a triangular prism with an angle slightly larger than the critical angle (around 61 degree at the glass/water interface) for total internal reflection illumination. The scattered signal generated by the GNR was collected by a 100x objective (NA 1.3, Olympus) and then projected onto a CMOS camera. The plasmonic probe in this study was synthesized via a seed-mediated process as described detailedly in the supplementary materials. Figure 2 shows the TEM result of the GNRs. The particles displayed well-defined rod shape with size around 60×19 nm. From the color-coded dark-field image under white light illumination, the majority of particles adsorbed on the glass slide surface exhibited evenly distributed red color, indicative of uniform size distribution and good colloidal stability. Since the dimension of the plasmonic nanoparticle is far smaller than the wavelength of the excitation light, the induced scattering response by an external electromagnetic field can thus be simplified into oscillations from point-like dipoles. Typically, for a nanorod, the scattering electrical field can be quantified by a linear superposition of three independent scattering electric fields associated with three mutually orthogonal dipoles.[7,8a,11] This is confirmed by the UV-vis absorption spectrum where two characteristic peaks at 530 nm and 631 nm were observed, corresponding to transverse oscillation mode along the short axes and longitudinal mode along the long axis, respectively. On this account, the spatial orientation of the nanorod under nanoscale environment could be determined readily by measuring the optical response from polarization dependent dipoles via either field distribution mapping or intensity characterization.[8b,11] Under normal TIRS mode with un-polarized light illumination, the PSF of the scattering image from individual GNR exhibits a well-defined Airy disk shape. This is because the unpolarized excitation light enables the induced scattering electromagnetic field from the nanorod span the whole space. In the farfield image plane, the collected lights were integrated into a diffraction-limited bright spot. The measured intensity distribution on the focal plane from a typical GNR is shown in Figure 2c. The

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Figure 2. a) UV-vis absorption spectrum and the corresponding TEM image (inserted) of GNRs. b) Color dark-field image of the GNRs on cover glass surface. c) The intensity profile of the TIRS image (inserted) from a single GNR with un-polarized light illumination. The dot curve is the measured result, which can be fitted with a simple Gaussian function (blue line). d) The corresponding polarization dependent scattering intensity curve of the GNR.

intensity profile of the cross line through the center of the bright spot in the TIRS image could be approximated as a normal Gaussian distribution function. It is thus a grand challenge to decode the full 3D angular information basically according to a single scattering image even though the observed nanoparticle is indeed from a single GNR as confirmed by polarization modulation measurement, Figure 2d. The measured scattering intensity is strongly dependent on the polarization direction of the incident light and can be well fitted with a squared cosine function    ∝ ∗    , where  is the amzimuthal angle of the GNR projected onto the x-y plane (or the angle between the optical axis of the polarizer and the long axis of the nanorod),  is the rotated angle of the polarizer relative to its optical axis,  is the measured intensity of the nanorod, and is a system dependent constant. In order to capture the angular dependent field distribution pattern from a single microscopic image, a degenerated PSF for the target object is normally required.[11,13] One of the commonly adopted method is to record the signal out of the focal plane where the electromagnetic field in the far field image plane is not well focused, resulting in angular dependent unique intensity distribution pattern. Interestingly, with TIRS illumination, when the excitation light was turned to p-polarization, the scattering image of the GNR was changed and degenerated into a doughnut shape. The orientation of the intensity distribution pattern differs from one to one and dependents on the orientation of the GNR, Figure 3. Further inspection of the TIRS image, interestingly, the pattern of the measured scattering image from this illumination mode is comparable to the previously observed defocused image from a conventional dark-field microscope.[11] It should be noted that the particles are still located in the focal plane when switched to ppolarized TIRS imaging mode. No aberration was introduced during the polarization modulation. On the contrary, when the polarization direction of the light was turned to s-polarization, the shape of the diffraction-limited PSF was returned again into a well-focused Airy disk (Gaussian shape), analogous to a normal dark-field image. The size of this focused Airy disk is smaller than the degenerated doughnut PSF. A set of images from a

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doughnut shape to a well-focused Airy disk by changing the polarization direction of the illumination light is shown in Figure 3c. The doughnut-shaped PSF is gradually constrained into a Gaussian PSF from p- to s-polarization illumination. The darkness center of the doughnut-shaped PSF is overlapped with the bright center of the Gaussian PSF. The appearance of distinct field distribution pattern in the scattering image under different polarization illumination can be clarified by taking account of the resulting electromagnetic field from the GNR induced by the polarized excitation light. As sketched in Figure 1, under s-polarization illumination, the oscillation direction of the excitation electric field is parallel to the solid/liquid interface. The induced electromagnetic fields from the GNR are essentially parallel to the glass slide surface. Therefore, the direction of the scattered light will transport toward the direction of the optical axis of the microscope, resulting well-focused PSF comparable to a regular dark-field image in the far-field image plane. It is thus difficult to elucidate the angular dependent scattering information from the well-focused image as noted above. While with p-polarization illumination, the evanescent field is a combination of strong vertical (z) and weak horizontal (x) components. In this case, the induced scattering light from individual GNR is mainly caused by the transverse oscillation electric field normal to the interface. The light emanating from the GNR therefore has components with an orientation parallel to the optical axis (normal to the solid/liquid interface) of the microscope and is also associated with the orientation of the GNR. The collected light in the image plane will then display in a characteristic doughnut shape owing to the spatial filtering of the collected scattered light.[14] The scenario described above was further confirmed by computer simulation based on the finite-difference time-domain method. As indicated in Figure1, for a GNR located at the solid/liquid interface with the polar angle of 10o and azimuthal angle of 90o, the electric field distribution pattern is indeed closely correlated with the polarization mode of the excitation light. Under s polarization illumination, the electric field exhibits a spherically symmetric shape with the peak located at the origin when observed in a direction normal to the interface. On the contrary, with p-polarized light illumination, the electric field is asymmetrically distributed in an orientation dependent manner. Until now, orientation dependent doughnut-shaped PSF of GNR can be achieved with un-polarized light through either defocused illumination under normal dark-field microscope or TIRS illumination on a thin metal film as far as we know.[11,14,15] In the former case, the strategy has been adopted to track the rotational dynamics of individual GNR in solution and on cell membrane.[11,16] Rotational dynamics of transferrin functionalized GNRs were tracked with high temporal resolution (50 Hz). However, one has to carefully manipulate the focal plane during the single particle tracking process because the pattern of the defocused image is closely associated with the defocusing distance of the probe relative to the focal plane. For the latter case, the resulted doughnut-shaped PSF is owing to the damped and enhanced horizontal and vertical dipolar response on a metal film, respectively. It is worthy of note that a thin metal film is a key requirement for the generation of orientation dependent doughnut-shaped PSF. The pattern of PSF is also strictly dependent on the distance between the nanoparticle and metal film because the coupling effect will be vanished once the distance was larger than tens of nanometers.[14] Furthermore, because of the strong energy transfer capability of the metal film, the fluorescence of dyes or nano-

Figure 3. a) and b) are typical TIRS images of GNRs with s- and ppolarized light illumination respectively. c) The polarization dependent (from s to p polarization) TIRS images from a single GNR (marked with a green circle in a) and b)).

materials close to surface could be quenched completely. It is therefore not convenient to extend this strategy to chemical or biological applications and combine with other imaging modality. To further explore the capability of this method for dynamic process tracking, as a proof of concept experiment, we trapped GNRs at the interface by negatively charged poly(acrylic acid) (PAA, MW:2000). The coverslip was firstly treated with 3Aminopropyltrimethoxysilane. The amino group functionalized coverslip exhibits a strong positive charge and is able to adsorb a thin layer of negatively charged PAA molecules. Then the GNRs were trapped on the polymer layer via electrostatic interaction. Owing to the loose and flexible net structure of the polymer layer, the nanoparticles on the surface exhibited very slow or even frozen translational motion. However, the rotational movement remains observable. A set of representative images from three vigorous rotating GNRs on the coverslip is illustrated in Figure 4. The pattern of TIRS image varies from each other. Through fitting the image with the simulated image (see Supporting Information),[11] the rotational dynamics of GNRs in the vertical and horizontal directions were revealed in Figure 4 with a time resolution of 90 Hz. The rapid fluctuations in polar and azimuthal angles as a function of time indicate that the GNRs were loosely trapped by the polymer. As the in-plane and out-of-plane rotational dynamics were revealed, it is thus capable to compare the rotational degrees of freedom in in-plane and out-of-plane by calculating the corresponding angular autocorrelation function separately, which indicates the time it takes for a particle to lose memory of its initial spin orientation.[17] Basically, the rotational diffusion of a GNR in a homogeneous environment can be simplified as a random walk of a unit vector on the surface of a unit sphere. The resulting rotational dynamics in different directions (i.e. x, y, z) under stationary condition are identical for isotropic rotational diffusion and the corresponding angular autocorrelation functions in these directions would be similar and exhibit a single exponential decay.[18] However, under a heterogeneous environment, the local viscosity of the system would significantly affect the rotational dynamics of the probe in different directions. Under this condition, characterizing the rotational behavior of the probe based on the time dependent polarization anisotropy track is no more a justified strategy. For example, the rocking process along z direction is hardly distinguishable from the polarization anisotropy. On this account,

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Figure 4. Rotational dynamics of three representative GNRs at the liquid/solid interface. From left to right are the time dependent polar (red) and azimuthal (green) angles of three GNRs. The corresponding orientational correlation functions (OCF) in in-plane and out-of-plane are shown in the right side respectively.

revealing the orientation dynamics in a 3D manner can provide more detailed and accurate dynamic rotational information. The autocorrelation decay curves of in-plane and out-of-plane angles from these GNRs are shown in Figure 4. For GNR 1, the autocorrelation functions in these two directions decay in a similar way, indicative of comparable rotational velocity in 3D. However, for GNR 2 and GNR 3, notable differences in the autocorrelation decay curves from the in-plane and out-of-plane directions are observable, demonstrating distinct friction forces between these two directions. For instance, there might be single or multiple anchoring points on the sidewall of the GNR, enabling restricted rotational diffusion at the interface. This kind of heterogeneous rotational diffusion is commonly observed for anisotropic nanoparticles on two-dimensional fluidic membrane such as cellular membrane. The dynamic ligand-receptor interactions could significantly affect the translational and rotational dynamics of the nanoparticle on the lipid membrane. As a consequence, the methodology presented here demonstrates a promising microrheological method that can be implemented to fluid membranes, including membranes with bound proteins.[19] In conclusion, we demonstrated a new and convenient method to resolve the angular information of individual GNR at the solid/liquid interface. The 3D angular information of a single GNR could be readily elucidated based on the field distribution pattern in a single TIRS image under p-polarization illumination. Distinct from the previously reported defocused dark-field illumination, the method presented herein doesn’t require complicated focal plane correction, affording a versatile pathway to study the rotational dynamics at the interface in a high-throughput manner. More importantly, under TIRS imaging mode, one could readily switch the image pattern of the probe between angle-coded degenerated PSF and position dependent Gaussian PSF for either angular determination or high accuracy spatial localization estimation simply by changing the polarization direction of the incident light, respectively. It could therefore be extensively applied in biology, chemistry and physics for static and dynamic exploration of angular information of rod shape nanoparticles in nanoscale system.

ASSOCIATED CONTENT Keywords: orientation tracking • total internal reflection • scattering imaging • gold nanorod • liquid/solid interface Supporting Information. The Supporting Information including details about experimental section is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author [email protected] Author Contributions †These authors contributed equally.

ACKNOWLEDGMENT This work was supported by NSFC (21205037, 21405045, 21522502), Program for New Century Excellent Talents in University (China, NCET-13-0789), Hunan Natural Science Funds for Distinguished Young Scholar (14JJ1017).

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A new strategy, total internal reflection scattering (TIRS) microscopy, was introduced to determine the full three-dimensional (3D) angular information of single gold nanorod (GNR) close to the liquid/solid interface. It was found that the 3D orientational information of individual GNR could be readily elucidated by using p-polarized TIRS illumination through deciphering the orientation-coded intensity distribution pattern in single TIRS image. In comparison with the previously reported strategies, this method doesn’t require complicated focal plane correction, affording a versatile pathway to track the rotational dynamics close to the interface in a high throughput manner.

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